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Sequencing

As a starting point we strongly suggest that users read sequencing guide . We also recommend that users follow our Sanger sequencing reaction protocol.

The table below shows the recommended quantity of template to use in a sequencing reaction (extracted from the DNA Sequencing by Capillary Electrophoresis by Applied Biosystems).

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TEMPLATERECOMMENDED QUANTITY
PCR Product:
100 - 200bp
200 - 500bp
500 - 1000bp
1000 - 2000bp
>2000bp

1 - 3 ng
3 - 10ng
5 - 20ng
10 - 40ng
40 - 100ng
Single-Stranded DNA50 - 100ng*
Double-Stranded DNA (Plasmid DNA)200 - 500ng*
Large DNA (e.g. BACs,PACs,YACs,cosmids and fosmids)0.5 - 1.0ug
Bacterial genomic DNA2 - 3ug

*The AB3730 DNA Analyzer is a highly sensitive instrument, you may not need this much template.

NOTE:

  • In general, higher DNA quantities give higher signal intensities, however if the quantities below are exceeded problems will occur with sequencing.
  • The template quantities stated below should work with all primers. You may be able to use less DNA, especially when sequencing with the -21 M13 primer.

 

Sequencing Guides

Below are links to resources for general information on Sanger sequencing and troubleshooting your data:

Ramaciotti Centre Sequencing Guide

DNA Sequencing by Capillary Electrophoresis by Applied Biosystems

Qiagen Guide to Template Purification and DNA Sequencing

 

Sequencing Clinic

The Centre runs a sequencing clinic whereby users of our service can book one on one time with a Sanger sequencing expert. Please contact us for further information or to make an appointment.

 

Fragment Analysis

The most common issue with fragment analysis is that the size standard does not run properly (no size data) or under/over-saturation of the fluorescence signals. Good sample preparation for fragment analysis requires a fine balance between the sample and the LIZ size standard, and if you are multiplexing, between all of the fluorescence labels and the size standard. Through proper optimisation, the data will produce clean, beautiful peaks for subsequent analysis.

Excess reagents and salt can interfere with the electrokinetic injection of the machine and lower the quality of the data. Other contaminants, such as residual protein or detergents carried over from DNA extraction can adhere to the capillaries of the array, thereby adversely reducing the quality of the data and life span of the array. If considerable dilution of the sample is not possible we require our customers to perform a clean-up step (e.g. ethanol precipitation) prior to submission.

Very strong signals are difficult to analyse and can cause similar negative impacts on the array as other contaminants so it is suggested that a 1:20 to 1:150 dilution series is completed on a small number of samples to optimise prior to submitting large numbers of samples. If size ranges do not overlap within a dye colour PCR products may be pooled or multiplex PCR used.

 

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